Preparation of protective antigen

ABSTRACT

A polynucleotide sequence is provided comprising a nucleic acid sequence encoding recombinant Protective Antigen (rPA). 
     Also provided are expression vectors and host cells comprising the polynucleotide sequence of the invention, and methods for producing rPA.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional application of co-pending U.S.application Ser. No. 12/042,150, filed Mar. 4, 2008, which is acontinuation-in-part application of U.S. application Ser. No.11/153,865, filed Jun. 15, 2005, entitled “BACILLUS ANTHRACIS PROTECTIVEANTIGEN”, (now U.S. Pat. No. 7,355,027), which claims the benefit ofU.S. Provisional Application 60/579,687, filed Jun. 16, 2004, entitled“PREPARATION OF PROTECTIVE ANTIGEN”.

SEQUENCE LISTING INCORPORATION BY REFERENCE

A sequence listing in an ASCII text file, having the name“MSQ01-010-DIVX-US_SEQUENCE_LISTING_(—)9-26-2011.txt”, created on 26Sep. 2011, and having a size of 307,748 bytes, is hereby incorporated byreference in its entirety.

The present invention relates to polynucleotides and vectors encodingrecombinant Bacillus anthracis protective antigen (rPA), methods ofproducing rPA, and uses thereof in antigenic compositions, such asvaccines.

Bacillus anthracis is a Gram positive, spore-forming bacterium and thecausative agent of anthrax. Anthrax is a disease of domestic and landanimals, and can affect humans through contact with infected animalproducts. In the lungs, anthrax can cause massive fluid build-up, tissuedecay, toxic shock and death.

Anthrax vaccine has been manufactured by the present Applicant for over40 years and, since 1979, has been the subject of a UK Product Licence(PL1511/0037) held by the Secretary of State for Health. However, withinthat time there has been little product development or advance in itsmanufacturing process.

The above vaccine preparation is now described in more detail. Culturesof the toxigenic, non-capsulating B. anthracis 34F2 “Sterne” strain [seeSterne, M. (1939). Onderstepoort J. of Veterinary Science and AnimalIndustry, 13, pp 307-312] are grown in multiple 500 mL volumes in apartially defined medium in Thompson bottles at 37° C. until the pH ofselected culture bottles falls below pH 7.4.

At the end of the growth period (approximately 24-28 hours) the culturesare harvested by aspiration, and the pooled supernatant fluidssterilised by filtration. Potassium aluminium sulphate solution isadded, and the resulting solution mixed. The pH is then adjusted to5.8-6.2, and the resulting flocculant (‘alum-precipitation’) allowed tosettle under gravity for up to one week at 5° C.

The precipitate is then concentrated 20-fold (by volume) by aspiration,and diluted 1:4 with a saline solution to provide a ‘5-fold’ concentrateof anthrax vaccine precipitate (AVP). This is the antigenic compositionthat is used for vaccine formulation. Although the vaccine is subjectedto animal tests for potency and safety prior to human use, there is noseparate routine biochemical characterisation.

One further cell-free anthrax vaccine is available for human use. Thisvaccine is produced in the United States of America and is broadlysimilar to that available under PL1511/0037, except that a different B.anthracis strain is used and grown anaerobically. The process isfermenter-based, and the culture filtrate is absorbed on to an aluminiumhydroxide suspension.

Other available vaccines comprise live, attenuated spore suspensions.However, because of the inherent risks associated with attenuatedpathogens, these vaccines are usually restricted to non-human use.

Anthrax toxin consists of three distinct polypeptides known asprotective antigen (PA), oedema factor (EF), and lethal factor (LF). Thetoxin components act in specific binary combinations of PA and EF toform oedema toxin (ET), which causes tissue oedema, and of PA and LF toform lethal toxin (LT), which is lethal to laboratory animals and causeslysis of monocyte and macrophage cells. Lethal toxin is considered to bethe principal cause of anthrax-associated death as a consequence of itscytotoxic effects on peripheral macrophages and other cells.

PA acts as a target cell-binding moiety and, after a site-specificN-terminal activation by a cell-associated protease (furin),oligomerises and provides a high-affinity binding component for which EFand LF compete. Following binding of EF or LF to activated PA, theresulting ET or LT complexes become internalised by an acidic endosomecompartment, and the toxin factors EF and LF are thereby delivered intothe cytosol of the target cell.

EF is a calcium- and calmodulin-dependent adenylyl cyclase thatcatalyses the conversion of intracellular ATP to cAMP. EF is active in avariety of intracellular signalling pathways, and is thereby capable ofdisrupting a range of cellular processes.

LF is a Zn²⁺-dependent metalloprotease that cleaves and inactivates thedual specificity, mitogen-activated protein kinase kinases MAPKK/1 and2, MEK-1 and MEK-2, and probably other proteins.

A survey of in vitro or in vivo published data on anthrax vaccines forhuman use indicates the following:—

-   1. to date, all effective anthrax vaccines contain or produce PA    (i.e. either the 83 kDa pro-form, or its activated 63 kDa    derivative). In fact, the current dogma is that PA is necessary and    sufficient alone to produce an effective anthrax vaccine, and    efforts are underway to develop such a vaccine [see, for example,    Baillie, L. (2001), 91, pp 609-613];-   2. the non-capsulated, toxigenic live-spore vaccines effect a higher    degree of protection against all B. anthracis strains so far tested    than do the licensed cell-free vaccines [see Little, S. F. (1986)    Inf. and Immunol. vol. 52, No. 2, pp 509-512];-   3. the current cell-free vaccines are generally poorly defined and    may vary significantly in effectiveness on a batch-by-batch basis.    Accordingly, each batch must be individually tested for efficacy in    an animal model prior to human use;-   4. the current cell-free anthrax vaccine manufacturing process is    evaluated only on completion of the production process and packaging    of the final product. Thus, in the event that any one batch of    vaccine material should not meet the validation test criteria, the    contributing factors cannot be identified readily. Such factors may    differ between manufactured batches and the lack of understanding    exacerbates any difficulties encountered in the manufacturing    process;-   5. as a result of the poorly defined nature of current cell-free    vaccines, these vaccines may contain quantities of PA together with    LF and/or EF which, upon in vivo (or in vitro) activation of PA to    the 63 kDa form, may form LT and ET and exert adverse effects on the    recipient of the vaccine. Such vaccines may, of course, also contain    other B. anthracis proteins, both secreted and lysis products,    peptidoglycan, nucleic acid and carbohydrate, which may compromise    protective efficacy;-   6. the current cell-free vaccine compositions are highly variable in    terms of LF, PA, and EF concentrations, so much so that EF may be    absent from some preparations; and-   7. the current cell-free compositions are highly variable in terms    of total protein content. Thus, the concentration of toxin    components present in a given composition may vary significantly.    This, in turn, may affect efficacy and potential toxicity in humans.

Over the last few years there has been notable academic research in theanthrax field. Sharma et al. (1996) describe the expression of native PAfrom E. coli. The signal sequence of the outer membrane protein A (OmpA)was added to the 5′-end of the PA gene and allowed the purification ofthe protein from the E. coli periplasmic space. Further research hasallowed identification of the native binding sites and translocationdomain of PA [see Bhatnagas, R. (2001) Critical Rev. in Microbiol.,27(3), pp 167-200; and Batra, S. (2001) Biochem. and Biophys. Res.Comm., 281, pp 186-192]. Thus, the structure and binding/translocationdomains of PA have been well documented.

Recently, a second-generation “recombinant” anthrax vaccine has beenproposed by The Ohio State University Research Foundation [see WO01/45639; and Price, B. M. (2001) Inf. and Immun., vol. 69, No. 7, pp4509-4515]. The described vaccine is based on PA and LF, wherein the LFmolecule has been modified so as to be zinc metalloprotease negative.Thus, the described PA and LF components are fully capable of binding toone another to form an LT molecule, but the resulting LT molecule is notcytotoxic as there is no active zinc metalloprotease function presentwith the LF component.

Ahuja Nidhi et al., Biochem. and Biophys. Research Communications, Vol.287, No. 2, 21 Sep. 2001, pp 542-549, describes PA mutants havingimpaired oligomerization and their potential as vaccine candidates.

Batra Smriti et al., Biochem. and Biophys. Research Communications, Vol.281, No. 1, 16 Feb. 2001, pp 186-192 describes PA mutants having mutantresidues that may have a role in membrane insertion of PA and/ortranslocation of LF/EF into the cytosol.

WO 02/04646 describes PA polypeptide domains capable of producing animmune response. The PA polypeptide is produced in E. coli andaccumulates in the form of inclusion bodies.

DNA-based anthrax vaccine compositions are described in WO 2004/024067.The vaccine compositions contain anthrax nucleic acids that have beenmodified to optimise expression in a eukaryotic host—e.g. the patient towhom the vaccine composition is administered.

In view of the increasing threats of bio-terrorism and biologicalwarfare, there is a need for alternative anthrax vaccines, and forvaccines that address one or more of the above-identified problems.

Thus, according to a first aspect of the present invention, there isprovided a polynucleotide sequence comprising a nucleic acid sequencehaving at least 75% identity to SEQ ID NO: 1, wherein said nucleic acidsequence encodes recombinant Bacillus anthracis Protective Antigen(rPA); or a fragment of said nucleic acid sequence wherein said fragmentencodes a fragment of recombinant Bacillus anthracis Protective Antigen(rPA).

In this regard, SEQ ID NO: 1 represents a modified nucleic acid thatencodes rPA. The sequence of SEQ ID NO: 1 is approximately 70% identicalto the wild-type Bacillus anthracis nucleic acid sequence encoding PA,provided herein as SEQ ID NO: 2.

The present inventors have found that by modifying the wild-type PAnucleic acid sequence (SEQ ID NO: 2), expression levels of rPA proteinmay be significantly improved. Thus, the present invention relates tonon-natural nucleic acid sequences which encode for the rPA polypeptide.Particularly, the non-natural nucleic acid sequences are selected toincrease expression levels of rPA expressed in heterologous systems,such as heterologous bacterial systems, e.g. E. coli. Preferably, therPA polypeptide or fragment thereof, which is expressed from themodified, non-natural nucleic acid sequence (or fragment thereof) of theinvention, is expressed at a level that is at least 110%, at least 120%,at least 150%, at least 200%, at least 250%, at least 300%, at least400%, or at least 500% higher than that expressed from the wild-typenucleic acid sequence under equivalent conditions.

The polynucleotide of the invention comprises a nucleic acid sequence(or fragment thereof) that encodes rPA (or a fragment thereof). This rPAencoding nucleic acid sequence (or fragment thereof) is referred toherein as the rPA nucleic acid (or fragment thereof). Thus, thepolynucleotide of the present invention may comprise the rPA nucleicacid, plus other coding and/or non-coding sequences. By way of example,non-coding sequences that may be comprised in the polynucleotide of thepresent invention include promoter sequences andtranscription/translation initiation and termination sequences.

In this regard, the rPA nucleic acid sequence of the present inventionmay embrace a number of modifications, which result in the sametranslated amino acid sequence of the encoded polypeptide. Numerousfactors should be taken into account when modifying a nucleic acidsequence, for example, the degree of degeneracy available, codon usage,and predicted RNA secondary structure considerations. For example, manyamino acids are designated by more than one codon, due to the“degeneracy” of the genetic code. In more detail, alanine is coded forby 4 different triplets, and serine is coded for by 6 differenttriplets. This degeneracy allows for DNA base composition to vary over awide range without altering the amino acid sequence of the proteinencoded by the DNA.

The wild-type polypeptide sequence of Bacillus anthracis UM44 PA isprovided in SEQ ID NO: 5 (see also, Vodkin, M., et al., Cell, 34:693(1983); and Welkos, S., et al., Gene, 69(2): 287 (1988)).

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences may be then compared. When using asequence comparison algorithm, test and reference sequences are inputinto a computer, subsequent coordinates are designated, if necessary,and sequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percentage sequence identityfor the test sequence(s) relative to the reference sequence, based onthe designated program parameters.

Optimal alignment of sequences for comparison may be conducted, forexample, by the local homology alignment algorithm of Smith and Waterman[Adv. Appl. Math. 2: 484 (1981)], by the algorithm of Needleman & Wunsch[J. Mol. Biol. 48: 443 (1970)] by the search for similarity method ofPearson & Lipman [Proc. Nat. Acad. Sci. USA 85: 2444 (1988)], bycomputer implementations of these algorithms (GAP, BESTFIT, FASTA, andTFASTA—Sequence Analysis Software Package of the Genetics ComputerGroup, University of Wisconsin Biotechnology Center, 1710 UniversityAvenue, Madison, Wis. 53705), or by visual inspection [see CurrentProtocols in Molecular Biology, F. M. Ausubel et al., eds, CurrentProtocols, a joint venture between Greene Publishing Associates, In. andJohn Wiley & Sons, Inc. (1995 Supplement) Ausubel].

Examples of algorithms suitable for determining percent sequencesimilarity are the BLAST and BLAST 2.0 algorithms [see Altschul (1990)J. Mol. Biol. 215: pp. 403-410; and “www.ncbi.nlm.nih.gov” of theNational Center for Biotechnology Information].

In one embodiment of a polypeptide homology comparison, the identityexists over a region of the sequences that is at least 10 amino acids,preferably at least 20 amino acids, more preferably at least 35 aminoacids in length. In a preferred polypeptide homology comparison, theidentity exists over a region of the sequences that is at least 100amino acids, preferably at least 200 amino acids, more preferably atleast 350 amino acids in length.

The terms “peptide” or “polypeptide” throughout this specification aresynonymous with the term “protein”, and do not refer to a specificlength of the product. These terms may embrace post-translationalmodifications such as glycosylation, acetylation, and phosphorylation.

Reference throughout the present application to rPA polypeptides,polynucleotides and nucleic acids embraces fragments, variants andderivatives thereof. In particular, reference throughout the presentapplication to rPA polypeptides embraces fragments, variants andderivatives thereof that have a common antigenic cross-reactivity withwild-type Bacillus anthracis PA (SEQ ID NO: 5). Similarly, referencethroughout the present application to rPA polynucleotides and nucleicacids embraces fragments, variants and derivatives thereof that encodepeptides having a common antigenic cross-reactivity with wild-typeBacillus anthracis PA (SEQ ID No. 5).

In one embodiment, the above-mentioned fragments, variants andderivatives may have a common antigenic cross-reactivity with one ormore of the four domains of the mature 735 amino acid monomer (see SEQID NO: 5) described below:

DOMAIN 1: amino acids 1-258. This domain binds two Ca²⁺ ions and is thecleavage site for proteases to activate the PA protein. The product ofthis cleavage is the amino terminal fragment a20 (20K fragment). A furincleavage site is located at amino acids 164-167.

DOMAIN 2: amino acids 259-487. This domain is involved in the formationof hexamer and has flexible loop which aids membrane insertion.

DOMAIN 3: amino acids 488-595. This domain currently has no knownfunction.

DOMAIN 4: amino acids 596-735. This domain is involved in receptorbinding.

In preferred embodiments, polypeptide “fragments” of the inventioncomprise at least one of the four domains identified above. Morepreferably, they comprise at least two, at least three, or all four ofthese domains in any combination. In a particular embodiment, theycomprise at least domains 2 & 3 identified above.

Each of the four domains identified above is considered to compriseimportant epitope(s) of wild-type Bacillus anthracis PA. In addition, PAepitopes have been identified as shown in the two tables below (the “BCell” table and the “T Cell” table).

In a preferred embodiment of the invention, polynucleotides are providedthat encode one or more epitopes or partial epitopes of PA. By way ofexample, SEQ ID NOs: 36-105 encode all or part of the first, and thethird to the sixth, epitopes listed in the “B Cell” table, and all threeof the epitopes listed in the “T Cell” table. SEQ ID NOs: 66-105 furtherencode the second epitope listed in the “B-Cell” table.

B-Cell Epitopes from Human and Epitope Non Human Primates SNHPs PositionImmunized in rPA Species Epitope Sequence protein Reference H. sapiensIKLNAKMNILIRDKRFHYDRN 581-601Les Baillie et al., “Characterisation of the human immune(SEQ ID NO: 107) response to the UK anthrax vaccine”, FEMS Immunol. Med.Microbiol. 2004 M.. PLYISNPNY  686-694Laffly et al., “Selection of a macaque Fab with framework fascicularis(SEQ ID NO: 108)regions like those in humans, high affinity, and abilityto neutralize the protective antigen of Bacillus anthracisby binding to the segment of PA between residues 686 and694”, Antimicrob. Agents Chemother. 2005 M. mulatta — 488-735 E D Williamson, et al. Infect. Immun. 2005 M. mulatta — 596-735 ″M. mulatta —   1-258 ″ P. — 614-735Chen et al., Efficient neutralization of anthrax toxin by troglodyteschimpanzee monoclonal antibodies against protectiveantigen, J. Infect. Dis. 2006

T-Cell Epitopes from Human and  Epitope Non Human Primates (NHPs)Position Immunized In rPA Species Epitope Sequence protein ReferenceH. sapiens PIYNVLPTTSLVLGKNQTLAT 373-393Laughlin et al., Antigen-specific CD4+ T cells (SEQ ID NO: 109)recognize epitopes of protective antigen following vaccination, Infect. Immun. 2007 H. sapiens SLVLGKNQTLAT 381-392 ″ (SEQ ID NO: 110) H. sapiens RLYQIKIQYQRENPTE  ″(SEQ ID NO: 111) 112-127

The term “fragment” of a polypeptide means a peptide consisting of atleast 5, preferably at least 10, more preferably at least 20, and mostpreferably at least 35 amino acid residues of the full-lengthpolypeptide that is the product of the polynucleotide in question. Thefragment preferably includes at least one epitope of the correspondingfull-length polypeptide. The fragment may result from enzymaticbreak-down of the corresponding full-length polypeptide. Alternatively,a fragment of the corresponding full-length polypeptide may be producedby expressing a polynucleotide that is fragment of the correspondingfull-length polynucleotide.

In preferred embodiments, the polypeptide “fragment” has an amino acidlength which is at least 10%, preferably at least 20%, preferably atleast 30%, preferably at least 40%, preferably at least 50%, preferablyat least 70%, and more preferably at least 80%, or at least 90%, that ofthe length of the amino acid sequence of the corresponding full-lengthpolypeptide. For example, the polypeptide fragment may comprise at least100, preferably at least 150, preferably at least 200, preferably atleast 300, most preferably at least 400, or at least 500, or at least600, or at least 700, amino acid residues, of the wild-type PApolypeptide sequence (SEQ ID NO: 5).

The present invention embraces “variants”. An example of a “variant” isa peptide or peptide fragment that contains one or more analogs of anamino acid (e.g. an unnatural amino acid), or a substituted linkage. Ina further embodiment, a “variant” may be a mimic of the peptide orpeptide fragment, which mimic reproduces at least one epitope of thepeptide or peptide fragment. The mimic may be, for example, a nucleicacid mimic, preferably a DNA mimic.

The present invention also embraces “derivatives”, meaning a proteincomprising the peptide (or fragment, or variant thereof) in question.Thus, a derivative may include the peptide in question, and a furtherpeptide sequence that may introduce one or more additional epitopes. Thefurther sequence should preferably not interfere with the basic foldingand thus conformational structure of the peptide in question.

Examples of a “derivative” are a fusion protein, a conjugate, and agraft. Thus, two or more peptides (or fragments, or variants) may bejoined together to form a derivative. Alternatively, a peptide (orfragment, or variant) may be joined to an unrelated molecule (e.g. asecond, unrelated peptide). Derivatives may be chemically synthesized,but will be typically prepared by recombinant nucleic acid methods.Additional components such as lipid, and/or polysaccharide, and/orpolyketide components may be included in a derivative.

All of the molecules “fragment”, “variant” and “derivative” have acommon antigenic cross-reactivity and/or substantially the same in vitroor in vivo biological activity as the product of the polynucleotide inquestion from which they are derived. By way of example, an antibodycapable of binding to a fragment, variant or derivative would be alsocapable of binding to the product of the polynucleotide in question.

It is a preferred feature that the fragment, variant and derivative eachpossess the active site of the peptide in question. Alternatively, allof the above embodiments of a peptide of the present invention share acommon ability to induce a “recall response” of a T-lymphocyte which hasbeen previously exposed to an antigenic component of a Bacillusanthracis infection.

An rPA peptide fragment, variant or derivative preferably has one ormore of the following properties—a) able to bind to the PA receptor on acell membrane; b) able to bind to EF and/or LF; and c) able to becleaved by furin protease. Thus, in one embodiment, a fragment, variantor derivative of a peptide of the present invention may be identified bycarrying out simple tests for the above-mentioned properties, asdescribed in WO 03/037370 which is incorporated by reference herein.

The terms DNA “fragment”, polynucleotide “fragment” and nucleic acid“fragment” used in this application refer to a polynucleotide that willusually comprise at least about 5 codons (15 nucleotides), more usuallyat least about 7 to 15 codons, and most preferably at least about 35codons. This number of nucleotides is usually about the minimal lengthrequired for a successful probe that would hybridize specifically (e.g.under selective hybridization conditions) with such a sequence.

Preferably, the DNA “fragment” of the invention comprises nucleotidesencoding at least one of the four PA protein domains identified above.More preferably, the DNA “fragment” comprises nucleotides encoding atleast two, at least three, or all four of the domains identified abovein any combination. The corresponding DNA base numbering for the four PAprotein domains is as follows:

DOMAIN 1: by 1-774 (774 bp)

DOMAIN 2: by 775-1461 (687 bp)

DOMAIN 3: by 1462-1785 (324 bp)

DOMAIN 4: by 1786-2205 (420 bp)

In a particular embodiment, the DNA “fragment” comprises DNA encodingprotein at least domains 2 & 3 as identified above. Examples of suchfragments are given in SEQ ID Nos: 36-105, which relate to truncatedversions of SEQ ID NO: 1. These truncated sequences encode domains 2 & 3in their entirety and substantial portions of both domains 1 & 4.

In preferred embodiments, the DNA “fragment” has a nucleotide lengthwhich is at least 10%, preferably at least 20%, preferably at least 30%,preferably at least 40%, preferably at least 50%, preferably at least70%, and more preferably at least 80% or at least 90% that of the codingsequence of the corresponding gene. For example, the fragment maycomprise at least 200, 300, 400, 500 or 600, preferably at least 900,most preferably at least 1200, or at least 1500, or at least 1700, or atleast 1900, or at least 2100 nucleotides of the full-length rPA nucleicacid sequence of the present invention. In particular embodiments, theDNA fragments have at least 1755, or at least 1806, or at least 1854, orat least 1857, or at least 1905, or at least 1953, or at least 2055nucleotides of the full-length rPA sequence of the present invention.

The present invention embraces DNA “variants”. A DNA variant is a DNAsequence that has substantial homology or substantial similarity to areference sequence, such as the coding sequence (or a fragment thereof)of the corresponding wild-type (natural) gene. A nucleic acid orfragment thereof is “substantially homologous” (or “substantiallysimilar”) to another if, when optimally aligned (with appropriatenucleotide insertions or deletions) with the other nucleic acid (or itscomplementary strand), there is nucleotide sequence identity in at leastabout 60% of the nucleotide bases, usually at least about 70%, moreusually at least about 80%, preferably at least about 90%, and morepreferably at least about 95 to 99% of the nucleotide bases. Homologydetermination is performed as described supra for peptides.

Alternatively, a DNA “variant” is substantially homologous (orsubstantially similar) with the coding sequence (or a fragment thereof)of a wild-type (natural) gene when it is capable of hybridizing underselective hybridization conditions. Nucleic acid hybridization will beaffected by such conditions as salt concentration (e.g. NaCl),temperature, or organic solvents, in addition to the base composition,length of the complementary strands, and the number of nucleotide basemismatches between the hybridizing nucleic acids, as will be readilyappreciated by those skilled in the art. Stringent temperatureconditions are preferably employed, and generally include temperaturesin excess of 30° C., typically in excess of 37° C. and preferably inexcess of 45° C. Stringent salt conditions will ordinarily be less than1000 mM, typically less than 500 mM, and preferably less than 200 mM.The pH is typically between 7.0 and 8.3. However, the combination ofparameters is much more important than the measure of any singleparameter. See, for example, Wetmur and Davidson (1968) J. Mol. Biol.31:349-370.

Selectivity of hybridization exists when hybridization occurs which issubstantially more selective than total lack of specificity. Typically,selective hybridization will occur when there is at least about 65%homology over a stretch of at least about 14 nucleotides, preferably atleast about 70%, more preferably at least about 75%, and most preferablyat least about 90% (see, Kanehisa (1984) Nuc. Acids Res. 12: 203-213).The length of homology comparison, as described, may be over longerstretches, and in certain embodiments will often be over a stretch of atleast about 17 nucleotides, usually at least about 20 nucleotides, moreusually at least about 24 nucleotides, typically at least about 28nucleotides, more typically at least about 32 nucleotides, andpreferably at least about 36 or more nucleotides. In a preferredembodiment, the length of homology comparison is over a stretch of atleast about 170 nucleotides, usually at least about 200 nucleotides,more usually at least about 240 nucleotides, typically at least about280 nucleotides, more typically at least about 320 nucleotides, andpreferably at least about 360 or more nucleotides.

The present invention embraces DNA “derivatives”, meaning a DNApolynucleotide which comprises a DNA sequence (or a fragment, or variantthereof) corresponding to the coding sequence of the reference gene,e.g. the wild-type Bacillus anthracis PA gene, and an additional DNAsequence which is not naturally associated with the DNA sequencecorresponding to the coding sequence. The comments on peptidederivatives supra also apply to DNA “derivatives”. A “derivative” may,for example, include two or more coding sequences of an operon. Thus,depending on the presence or absence of a non-coding region between thecoding sequences, the expression product(s) of such a “derivative” maybe a fusion protein, or separate peptide products encoded by theindividual coding regions.

The above terms DNA “fragment”, “variant”, and “derivative” have incommon with each other that the resulting peptide products havecross-reactive antigenic properties, which are substantially the same asthose of the corresponding wild-type peptide. Preferably all of thepeptide products of the above DNA molecule embodiments of the presentinvention bind to an antibody which also binds to the wild-type peptide.Alternatively, all of the above peptide products are capable of inducinga “recall response” of a T lymphocyte, which has been previously exposedto an antigenic component of a Bacillus anthracis infection.

Thus, a DNA fragment, variant or derivative may be identified by way ofits encoded peptide product—for example, by carrying out the simpletests mentioned above (and described in WO 03/037370).

Polynucleotides of the present invention comprise a nucleic acid havingat least 75%, preferably at least 80%, more preferably at least 85%,more preferably at least 90%, even more preferably at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% and most preferably 99% or 100% identity toSEQ ID NO: 1; or a fragment of said nucleic acid.

Specific polynucleotides having between 91 and 99% identity to SEQ IDNO: 1 (minus the terminal “TAA” stop codon) are provided in SEQ ID NOs:9 to 35 as shown in the table below.

% Identity No. of bases to SEQ ID differing from SEQ ID NO No. 1 SEQ IDNo. 1 SEQ ID No. 9 99.00 22 SEQ ID No. 10 99.00 22 SEQ ID No. 11 99.0022 SEQ ID No. 12 98.00 44 SEQ ID No. 13 98.00 44 SEQ ID No. 14 98.00 44SEQ ID No. 15 97.01 66 SEQ ID No. 16 98.00 66 SEQ ID No. 17 98.00 66 SEQID No. 18 96.01 88 SEQ ID No. 19 96.01 88 SEQ ID No. 20 96.01 88 SEQ IDNo. 21 95.01 110 SEQ ID No. 22 95.01 110 SEQ ID No. 23 95.01 110 SEQ IDNo. 24 94.01 132 SEQ ID No. 25 94.01 132 SEQ ID No. 26 94.01 132 SEQ IDNo. 27 93.02 154 SEQ ID No. 28 93.02 154 SEQ ID No. 29 93.02 154 SEQ IDNo. 30 92.02 176 SEQ ID No. 31 92.02 176 SEQ ID No. 32 92.02 176 SEQ IDNo. 33 91.02 198 SEQ ID No. 34 91.02 198 SEQ ID No. 35 91.02 198

In one embodiment, polynucleotides of the invention comprise a nucleicacid having at least 75%, preferably at least 80%, more preferably atleast 85%, more preferably at least 90%, even more preferably at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and most preferably 99% or 100%identity to SEQ ID NO: 36. SEQ ID NO: 36 is a truncated version of SEQID NO: 1 and comprises base pairs 301-2055 of SEQ ID NO: 1. Specificpolynucleotides having between 91 and 99% identity to SEQ ID NO: 36 areprovided in SEQ ID NOs: 37 to 45 as shown in the table below.

% Identity No. of bases to SEQ ID differing from SEQ ID NO No. 36 SEQ IDNo. 36 SEQ ID No. 37 98.97 18 SEQ ID No. 38 98.06 34 SEQ ID No. 39 97.0452 SEQ ID No. 40 95.90 72 SEQ ID No. 41 94.99 88 SEQ ID No. 42 93.96 106SEQ ID No. 43 93.16 120 SEQ ID No. 44 92.02 140 SEQ ID No. 45 91.00 158

In another embodiment, polynucleotides of the invention comprise anucleic acid having at least 75%, preferably at least 80%, morepreferably at least 85%, more preferably at least 90%, even morepreferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and mostpreferably 99% or 100% identity to SEQ ID NO: 46. SEQ ID NO: 46 is atruncated version of SEQ ID NO: 1 and comprises base pairs 202-2055 ofSEQ ID NO: 1. Specific polynucleotides having between 91 and 99%identity to SEQ ID NO: 46 are provided in SEQ ID NOs: 47-55 as shown inthe table below.

% Identity No. of bases to SEQ ID differing from SEQ ID NO No. 46 SEQ IDNo. 46 SEQ ID No. 47 99.03 18 SEQ ID No. 48 97.95 38 SEQ ID No. 49 97.0954 SEQ ID No. 50 96.12 72 SEQ ID No. 51 95.04 92 SEQ ID No. 52 93.96 112SEQ ID No. 53 92.99 130 SEQ ID No. 54 92.13 146 SEQ ID No. 55 91.05 166

In another embodiment, polynucleotides of the invention comprise anucleic acid having at least 75%, preferably at least 80%, morepreferably at least 85%, more preferably at least 90%, even morepreferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and mostpreferably 99% or 100% identity to SEQ ID NO: 56. SEQ ID NO: 56 is atruncated version of SEQ ID NO: 1 and comprises base pairs 103-2055 ofSEQ ID NO: 1. Specific polynucleotides having between 91 and 99%identity to SEQ ID NO: 56 are provided in SEQ ID NOs: 57-65 as shown inthe table below.

% Identity No. of bases to SEQ ID differing from SEQ ID NO No. 56 SEQ IDNo. 56 SEQ ID No. 57 99.08 18 SEQ ID No. 58 97.95 40 SEQ ID No. 59 97.0358 SEQ ID No. 60 96.01 78 SEQ ID No. 61 94.98 98 SEQ ID No. 62 94.06 116SEQ ID No. 63 93.04 136 SEQ ID No. 64 92.01 156 SEQ ID No. 65 90.99 176

In another embodiment, polynucleotides of the invention comprise anucleic acid having at least 75%, preferably at least 80%, morepreferably at least 85%, more preferably at least 90%, even morepreferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and mostpreferably 99% or 100% identity to SEQ ID NO: 66. SEQ ID NO: 66 is atruncated version of SEQ ID NO: 1 and comprises base pairs 301-2106 ofSEQ ID NO: 1. Specific polynucleotides having between 91 and 99%identity to SEQ ID NO: 66 are provided in SEQ ID NOs: 67-75 as shown inthe table below.

% Identity No. of bases to SEQ ID differing from SEQ ID NO No. 66 SEQ IDNo. 66 SEQ ID No. 67 99.00 18 SEQ ID No. 68 97.90 38 SEQ ID No. 69 97.0154 SEQ ID No. 70 96.12 70 SEQ ID No. 71 95.02 90 SEQ ID No. 72 94.02 108SEQ ID No. 73 93.02 126 SEQ ID No. 74 92.03 144 SEQ ID No. 75 91.03 162

In another embodiment, polynucleotides of the invention comprise anucleic acid having at least 75%, preferably at least 80%, morepreferably at least 85%, more preferably at least 90%, even morepreferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and mostpreferably 99% or 100% identity to SEQ ID NO: 76. SEQ ID NO: 76 is atruncated version of SEQ ID NO: 1 and comprises base pairs 301-2157 ofSEQ ID NO: 1. Specific polynucleotides having between 91 and 99%identity to SEQ ID NO: 76 are provided in SEQ ID NOs: 77-85 as shown inthe table below.

% Identity No. of bases to SEQ ID differing from SEQ ID NO No. 76 SEQ IDNo. 76 SEQ ID No. 77 99.03 18 SEQ ID No. 78 98.06 36 SEQ ID No. 79 96.9856 SEQ ID No. 80 96.02 74 SEQ ID No. 81 95.05 92 SEQ ID No. 82 93.97 112SEQ ID No. 83 93.00 130 SEQ ID No. 84 92.03 148 SEQ ID No. 85 90.95 168

In another embodiment, polynucleotides of the invention comprise anucleic acid having at least 75%, preferably at least 80%, morepreferably at least 85%, more preferably at least 90%, even morepreferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and mostpreferably 99% or 100% identity to SEQ ID NO: 86. SEQ ID NO: 86 is atruncated version of SEQ ID NO: 1 and comprises base pairs 202-2106 ofSEQ ID NO: 1. Specific polynucleotides having between 91 and 99%identity to SEQ ID NO: 86 are provided in SEQ ID NOs: 87-95 as shown inthe table below.

% Identity No. of bases to SEQ ID differing from SEQ ID NO No. 86 SEQ IDNo. 86 SEQ ID No. 87 98.95 20 SEQ ID No. 88 98.01 38 SEQ ID No. 89 96.9658 SEQ ID No. 90 96.01 76 SEQ ID No. 91 94.96 96 SEQ ID No. 92 94.02 114SEQ ID No. 93 92.97 134 SEQ ID No. 94 92.02 152 SEQ ID No. 95 90.97 172

In another embodiment, polynucleotides of the invention comprise anucleic acid having at least 75%, preferably at least 80%, morepreferably at least 85%, more preferably at least 90%, even morepreferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and mostpreferably 99% or 100% identity to SEQ ID NO: 96. SEQ ID NO: 96 is atruncated version of SEQ ID NO: 1 and comprises base pairs 103-2157 ofSEQ ID NO: 1. Specific polynucleotides having between 91 and 99%identity to SEQ ID NO: 96 are provided in SEQ ID NOs: 97-105 as shown inthe table below.

% Identity No. of bases to SEQ ID differing from SEQ ID NO No. 96 SEQ IDNo. 96 SEQ ID No. 97 99.03 20 SEQ ID No. 98 97.96 42 SEQ ID No. 99 96.9862 SEQ ID No. 100 96.01 82 SEQ ID No. 101 95.04 102 SEQ ID No. 102 93.97124 SEQ ID No. 103 93.00 144 SEQ ID No. 104 92.02 164 SEQ ID No. 10591.15 182

In one embodiment, polynucleotide sequences of the present inventionfurther comprise a polynucleotide encoding a secretion sequence. Thepolynucleotide encoding the secretion sequence is preferably clonedupstream of the rPA nucleic acid sequence, or fragment thereof, and ismost preferably operatively linked to said nucleic acid sequence, orfragment thereof.

Secretion sequences may allow the encoded protein to cross and/or lodgein cell membranes, and thus attain its functional topology or besecreted from a host cell. In this regard, the secretion sequence may befor extracellular translocation of the expressed polypeptide from a hostcell (e.g. a bacterial host cell) into the extracellular environment.Alternatively, the secretion sequence may be for periplasmictranslocation of the expressed polypeptide from a bacterial host cellcytoplasm into the periplasmic space.

It is particularly preferred that the secretion sequence is cleavablefrom the expressed polypeptide during periplasmic translocation orduring extracellular translocation, in which case theperiplasmic/extracellular polypeptide is free of this sequence. Oneexample of such a ‘cleavable’ sequence is a cpg leader sequence encodedby SEQ ID NO: 3.

The cpg leader sequence illustrated in SEQ ID NO: 3 is designed suchthat it has a 5′-NdeI restriction site for inserting into an expressionvector at a NdeI site, and a 3′-MscI site for fusion with a rPA nucleicacid sequence, or fragment thereof, where a similar MscI site haspreferably been engineered. Thus, the cpg leader is cleaved from theexpressed rPA protein (or protein fragment) as it passes through a hostcell membrane, leaving the ‘trimmed’ mature rPA protein, or fragmentthereof, in the extracellular environment or periplasmic spaceaccordingly.

Other suitable secretion sequences for use in the present invention aredescribed in Watson (1984) Proc. Nat. Acad. Sci. USA. vol. 12: 5145; andMakrides (1996) Microbiological Reviews 60: 512-538; and include, ompA(Denefle et al. (1989) Gene 85: 4990-510; and Ghrayeb et al. (1984) EMBOJ. 3: 2437-2442.); pelB (Better et al. (1988) Science 240: 1041-1043;and Lei et al. (1987) J. Bacteriol. 169: 4379-4383); including adegenerate version thereof—see Le Calvez et al. (1996) Gene 170: 51-55;phoA (Denefle et al. (1989) Gene 85: 499-510; and Oka et al. (1985)Proc. Nat. Acad. Sci. USA. 82: 7212-7216); ompT (Johnson et al. (1996)Protein Expression Purif. 7: 104-1123); lamB (Hoffman & Wright (1985)Proc. Nat. Acad. Sci. USA. 82: 5107-5111); ompF (Hoffman & Wright(1985)); beta lactamase (Kadonaga et al. (1984) J. Biol. Chem. 259:2149-2154; and Villa-Komaroff et al. (1977) Proc. Nat. Acad. Sci. USA.75: 3727-3731); Staphylococcus aureus protein A (Abrahmsen (1986) Nucl.Acids Res. 14: 7487-7500; and Macyntyre & Henning (1990) Biochimmie 72:157-167); Bacillus subtilis endoglucanase (Proudfoot et al. (1996) J.Biol. Chem. 271: 2599-2603); murine RNAse (Schein et al. (1992) Biochem.J. 283: 137-144); human growth hormone (Gray et al. (1985) Gene 39:247-254); and enterotoxins ST-II, LT-A and LT-B (Fujimoto et al. (1988)J. Biotechnol. 8: 77-86; and Morioka-Fujimoto et al. (1991) J. Biol.Chem. 266: 1728-1732).

In one embodiment, the polynucleotide sequence of the present inventioncomprises a nucleic acid of the present invention, or a fragmentthereof, having a 3′ and a 5′ end, and said nucleic acid or fragmentthereof has a codon encoding a methionine residue cloned to the 5′ end.By way of example, the nucleic acid may be SEQ ID NO: 7 (or a fragmentof SEQ ID NO: 7 that includes the 5′ terminal, met-encoding codon of SEQID NO: 5). This methionine-encoding codon is added in order to increasestability of the final (signal-less) protein when expressed in E. coli.Thus, rPA protein encoded by the polynucleotide of this particularembodiment of the invention is distinguished from wild-type PA proteinnaturally produced in Bacillus anthracis by the addition of an extramethionine residue to the N-terminus.

The present invention thus also provides a polypeptide or polypeptidefragment encoded by the polynucleotide of the present invention.Polypeptides of the present invention may therefore comprise an aminoacid sequence encoding rPA, or a fragment thereof, with an extramethionine residue added at the N-terminus of the rPA amino acidsequence.

In a related aspect, the present invention also provides an isolated RNAmolecule that is encoded by a DNA polynucleotide sequence of the presentinvention, or a fragment or variant or derivative of said DNA sequence.

Also contemplated within the invention are expression vectors comprisinga polynucleotide of the present invention. Expression vectors are usefulfor the expression of heterologous nucleic acid sequences in a hostcell. As used herein, the term “heterologous” means that thepolynucleotide or polypeptide sequence concerned does not naturallyexist in the cell, but has been introduced into it, for example bytransformation, transfection, injection etc.

Expression vectors generally are replicable polynucleotide constructsthat include coding regions for a peptide, operably linked to suitabletranscriptional and translational regulatory elements. Examples ofregulatory elements usually included in expression vectors arepromoters, enhancers, ribosomal binding sites, and transcription andtranslation initiation and termination sequences. These regulatoryelements are operably linked to the sequence to be translated. A nucleicacid sequence is operably linked when it is placed into a functionalrelationship with another nucleic acid sequence. For instance, apromoter is operably linked to a coding sequence if the promoter affectsits transcription or expression. Generally, “operably linked” means thatthe DNA sequences being linked are contiguous and, where necessary tojoin two protein coding regions, contiguous and in reading frame. Theregulatory elements employed in the expression vectors containing apolynucleotide encoding a virulence factor are functional in the hostcell used for expression.

It is preferred that the expression vector expresses the polynucleotidein the absence of a chemical inducer—i.e. a chemical inducer is notrequired for induction of expression from the expression vector. In oneembodiment, the vector expresses the polynucleotide constitutively, withno induction of gene expression needed. In another embodiment, thevector expresses the polynucleotide in response to an environmentalstimulus or stimuli—such as starvation, or limitation of nutrients oroxygen, such as when a component or components become exhausted in thegrowth medium.

The term “promoter” is well known in the art, encompasses relativelysimple, minimal promoters to complex promoters having upstream elementsand enhancers. Suitable promoters for expression in prokaryotic andeukaryotic host cells are well known in the art, and are described in,for example, Molecular Cloning. A laboratory Manual (Sambrook et al.,Second edition, 1989) and Current Protocols in Molecular Biology(Ausubel et al., eds., 1994).

Appropriate promoter and other necessary vector sequences are selectedso as to be functional in the host. By way of example, promoters such asthe trp, lac and phage promoters (e.g. T7, T4, lambda, fd), tRNApromoters and glycolytic enzyme promoters may be used in prokaryotichosts. It is preferred that the expression vector comprises a “strong”promoter,—i.e. a promoter that is selected so as to ensure that theencoded rPA polypeptide (or fragment thereof) is highly expressed.Examples of strong promoters include recA, malate dehydrogenase, T7,tac, etc. In this regard, a polypeptide is said to be “highly expressed”if it is expressed at levels above 20% of total host cell solubleprotein, preferably above 30%, more preferably above 40% and mostpreferably above 50% total host cell soluble protein. A preferred“strong promoter” for use in accordance with the invention is the malatedehydrogenase (mdh) promoter (proprietary to CAMR; U.S. Pat. No.5,670,333).

Expression vectors may contain a selectable marker—i.e. a gene encodinga protein necessary for the survival or growth of a host celltransformed with the vector. The presence of this gene ensures thegrowth on a selective medium of only those host cells that contain thedesired vector and that express the selectable marker. Typical selectiongenes encode proteins that: (a) confer resistance to antibiotics orother toxic substances, e.g. ampicillin, tetracycline, neomycin,methotrexate, etc.; (b) complement auxotrophic deficiencies; or (c)supply critical nutrients not available from complex media. Theselection of an appropriate vector and an appropriate selectable markerwill depend on the host cell, and is well within the capabilities of anordinary person of skill in the art.

Expression vectors typically contain all of the additional elements thatare necessary for efficient expression of the nucleic acid in a hostcell. Examples of suitable vectors for expression of heterologousproteins in bacterial include pET vectors (for example pET26b-Novagen),and pTrKHis (Invitrogen). Both these vectors achieve high-levelexpression of nucleic acid in E. coli.

pMTL expression vectors are advantageous because they are capable ofyielding high levels of recombinant protein, and can be very stable,even in the absence of selective pressure using antibiotics.Furthermore, those pMTL vectors based on the very strong E. coli malatedehydrogenase (mdh) promoter are particularly advantageous becauseinduction of expression using exogenous inducer chemicals is notrequired (Alldread et al. (1992) Gene 14: 139-143). It is an advantagenot to require an exogenous chemical inducer for regulatory reasons,since any chemical inducer may have to be rigorously and successfullyremoved from the final product before administration to patients.

In a particularly preferred embodiment, the expression vector is also ahigh copy number plasmid, such as pMTL1015 (Chambers et al. (1988) Gene68: 139-149). pMTL1015 is a derivative of pMTL4, it replicates from amutant of the ColE1 replicon (600 copies per cell; Minton et al. (1988)Focus 10: 56) and is encoded by SEQ ID NO: 4. Plasmid pMTL1015 isessentially identical to pMTL1003 as described by Brehm et al. (1991)Appl. Microbiol Biotechnol 36: 358-363, and has numerous advantageousfeatures. By way of example, pMTL1015 differs from pMTL1003 in that thetrp promoter has been replaced with the very powerful mdh promoter(Alldread et al. (1992) Gene 14: 139-143), and the ampicillin resistancegene has been replaced with the tetracycline resistance gene of pBR322(Bolivar et al. (1977) Gene 2: 95). The plasmid also incorporates thepSC101 partition function (par; Miller et al. (1983) Gene 24: 309-315),the rrnB double terminator (Brosius et al. (1981) J. Mol. Biol. 148:107-127) and the pMTL20 polylinker cloning region (Chambers et al.,1988). The par locus endows the plasmid with good segregationalstability enabling antibiotic-free fermentations without plasmid lossand the tetracycline resistance marker is a biopharmaceuticallyacceptable drug.

An example of an expression vector suitable for use in the presentinvention is the vector deposited under ECACC No: 04061401.

An example of an expression vector comprising a polynucleotide accordingto the present invention is pMTL1015-cpg-PA-synt, deposited under ECACCNo: 04052501.

The vectors containing the nucleic acids of interest can be transcribedin vitro and the resulting RNA introduced into the host cell (e.g. byinjection), or the vectors can be introduced directly into host cells bymethods which vary depending on the type of cellular host, includingelectroporation; transfection employing calcium chloride, rubidiumchloride, calcium phosphate, DEAE-dextran, or other substances;microprojectile bombardment; lipofection; infection (where the vector isan infectious agent, such as a retroviral genome). The term “host cells”is meant to embrace the progeny of such cells.

The present application thus also provides a host cell comprising anexpression vector as described above. It is preferred that the host cellis a bacterial cell, in particular an E. coli cell, such as E. colistrains DHS, BL21 and HMS174 (Invitrogen).

It is particularly preferred that the bacterial cell e.g. E. coli strainis a protease-deficient strain, since rPA protein is generallyconsidered to be a protease-sensitive protein. One example of aprotease-deficient strain of E. coli is E. coli RV308 (ATCC No: 31608).

Also provided by the present invention are methods for producing rPAcomprising expressing the polynucleotide of the present invention.

In one embodiment, the polynucleotide is expressed—i.e. transcribed andtranslated, in a host cell. In another embodiment, the polynucleotide isDNA, which is transcribed into RNA in vitro, and then the RNA is thentranslated into protein in a host cell. The host cell may be a bacterialcell, such as an E. coli host cell. The E. coil host cell is preferablya protease-deficient strain, such as E. coli RV308 (deposited under ATCCNo: 31608).

In a preferred embodiment, rPA is expressed in a host cell from theexpression vector of the present invention, as described above. In thisembodiment, the method may incorporate at least one, preferably two,most preferably all of the following features:—(i) the expression vectorcomprises the cpg leader sequence encoded by SEQ ID NO: 3; (ii) thevector is the plasmid pMTL1015, encoded by SEQ ID NO: 4; and (iii) thevector is expressed in an E. coli host cell such as E. coli RV308 (ATCCNo: 31608).

In one embodiment, the method comprises the initial steps oftransforming an expression vector comprising the polynucleotide ofinterest into a host cell, such as E. coli host cell and culturing thetransformed host cell in a suitable growth medium.

Optionally, the culture is carried out under selective pressure, such asin the presence of an antibiotic, e.g. tetracycline, in which case it isan advantage for the expression vector to comprise a selectable markerthat confers resistance to the antibiotic.

Culture parameters may be controlled, in order to control nutrients, pHand/or oxygen levels (dissolved oxygen tension—DOT) in the culture. Forexample, DOT may be controlled by agitation, back pressure, spargedairflow and/or oxygen supplementation. It is preferred that DOT ismaintained at above 40%.

The temperature at which host cells are grown can have an effect on thelevel of protein that can be purified from the culture. For example,protein expression rate and protein degradation rate (such as due toprotease activity) can both affect the amount of protein that can beextracted. Growing the cultured host cells comprising the claimedpolynucleotide at a reduced temperature of less than, for example 40°C., has been found to give acceptable levels of rPA expression andstability. Thus, in one embodiment of the invention, host cellscontaining polynucleotides of the invention (e.g. expression vectors)are cultured at less than 40° C.; preferably at less than 37° C.; morepreferably at less than 35° C.; more preferably at about 30° C., andmost preferably at 25-30° C., such as 29° C., 28° C., 27° C., 26° C. and25° C. Culturing host cells at these reduced temperatures may slow downthe rate of rPA expression, but this may be useful if a high-levelexpression vector, such as the plasmid pMTL1015, is used for expression.

It is preferred that a growth medium is used that is free of animalproducts (i.e. products derived from animals), since this isadvantageous for meeting the regulations for injectable products.Examples of suitable media include phytone peptone-based Terrific Broth,and soy peptone-based L-broth.

If a secretion sequence is used that enables extracellular secretion ofthe polypeptide into the growth medium then the growth medium may beharvested and undergo further purification steps to extract thepolypeptide.

Alternatively, if the secretion sequence enables secretion of thepolypeptide into the bacterial periplasm then the polypeptide productwill be intracellular. In this case, the cells must be harvested fromthe culture medium (e.g. by centrifugation as a cell paste) and undergofurther processing to extract the polypeptide from the cells. Suitableprotocols for the harvesting of cell cultures, such as bacterialcultures, for the purification of polypeptides are well known in theart, and can be found in common laboratory manuals such as Sambrook etal. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, ColdSpring Harbor Laboratory Press; and Sambrook and Russell (2001)Molecular Cloning: A Laboratory Manual, Third Edition, Cold SpringHarbor Laboratory Press.

Typically, bacterial cells can be harvested by centrifugation forextraction of either nucleic acids or polypeptides. For proteinpurification the conditions selected for the harvesting of culturedcells by centrifugation are generally gentler than for the extraction ofnucleic acid, so as not to damage the target protein. For example, theharvesting of bacterial cells for extraction of a target polypeptide maybe carried out at 4° C., by centrifugation at 4,000-5,000 g for 10-15minutes.

It is an option for the method to further comprise testing steps, toidentify the presence and/or yield of desired polypeptide, prior tofurther processing. In one embodiment, an ELISA-based test is carriedout.

Following the fermentation (bacterial growth and harvesting) andoptional testing protocols, the method may further comprise downstreamprocessing steps in order to obtain isolated, purified, rPA protein.

The downstream processing steps employed in the present inventionpreferably achieve one or more of the following aims:—

-   -   reduction in the number of chromatography steps required,        compared to prior art methods;    -   use of step elution rather than gradient elution for some,        preferably all, chromatography steps;    -   increase in the level of primary processing prior to        chromatography, compared to prior art methods;    -   removal of the need for the addition of conditioning agents        (e.g. nucleases) where possible;    -   use of techniques capable of scaling-up to at least 100 L        fermentation scale; and    -   use of techniques that are compatible with cGMP.

It is preferred that the purification procedure has reduced processtimes and volumes and/or has increased process efficiency in comparisonto prior art methods. In the present invention, the number ofdialysis/buffer exchange steps is preferably minimised, for example, bylinking steps that generate a process stream of high conductivity withthose that require a high conductivity starting material (e.g. ammoniumsulphate precipitation or ion-exchange chromatography may be followed byhydrophobic interaction chromatography).

The downstream processing protocol commences with a crude mixturecontaining rPA polypeptide. If the rPA polypeptide is located within thehost cell (e.g. within a bacterial host cell periplasm) then the cellsmust be treated to extract the rPA polypeptide, for example byhomogenisation.

It is preferred that the method further comprises at least oneseparation step, carried out on the extracted rPA polypeptide. Examplesof separation steps that may be included in the method are filtrationsteps such as diafiltration steps, and chromatography steps. In oneembodiment, the method comprises at least one chromatography step and atleast one filtration step.

In a particularly preferred embodiment of the present method, theextracted rPA polypeptide (together with unwanted components such asnucleic acids, other proteins, and cell debris) is subjected todiafiltration, such as tangental flow diafiltration. The purpose of thisstep is to alter the load of charged molecules, in preparation forsubsequent separation steps, such as chromatography steps. Diafiltersretain molecules of above a certain molecular weight (e.g. above 30 kDa,40 kDa or 50 kDa) and allow dissolved substances and those below thespecified molecular weight to pass through the filter. Thus, it ispreferred that the method includes at least one filtration step that isa diafiltration step.

Chromatography steps may include ion-exchange chromatography (e.g. usinga Q-sepharose anion exchange column) and hydrophobic chargechromatography (e.g. using a mercaptoethyl pyridine hypercel column).Other examples of suitable chromatographic techniques are known in theart and would be routinely available to a skilled person. Thus, thepresent method may include at least one ion-exchange chromatography stepand at least one hydrophobic charge chromatography step.

In one embodiment, when the rPA polypeptide has been expressed in an E.coli host cell, there may be residual E. coli endotoxin associated withthe rPA polypeptide and this can be separated from the rPA polypeptideby a (further) separation step, if necessary. In one embodiment,separation of endotoxin may be achieved by filtration, using a chargedfilter to which the toxin adheres.

Thus, in a specific embodiment, a method of producing rPA comprises thesteps of obtaining host cells that express the polypeptide of thepresent invention; extracting the expressed rPA from the host cells;subjecting the extracted rPA to a diafiltration step (e.g. tangentalflow diafiltration at 30 kDa); followed by at least one chromatographystep selected from ion exchange chromatography and hydrophobic chargechromatography; then a further diafiltration step (which may be at ahigher molecular weight cut-off e.g. 40 kDa or 50 kDa); and an optionalfurther filtration step to remove any residual protein and/or bacterialendotoxin.

In one embodiment of the present invention, the combination ofhigh-level gene expression (plasmid containing strong promoter),periplasmic translocation (secretion sequence), nucleic acid sequencemodification (rPA nucleic acid sequence) and efficient downstreamprocessing, results in an increase of rPA protein yields that are 10 to20-fold above yields previously available in the prior art.

Furthermore, the downstream processing steps of the present inventionallow rPA protein to be obtained that has greater than 70%, preferablygreater than 80%, greater than 90%, or greater than 95%, and morepreferably greater than 98% purity.

Polypeptide purity or homogeneity may be indicated by, for example,polyacrylamide gel electrophoresis of a protein sample, followed byvisualizing a single polypeptide band upon staining the gel.Alternatively, higher resolution may be provided by using, for example,HPLC.

If desirable, the amino acid sequence of the polypeptides of the presentinvention may be determined by protein sequencing methods.

The present invention thus also provides an rPA polypeptide or fragmentthereof produced by the method of the present invention. In oneembodiment, the polypeptide may be identical to wild-type PA produced byBacillus anthracis. In another embodiment, as described above, thepolypeptide or fragment thereof may be distinguished from wild-type PA(or a fragment thereof) by the presence of an extra residue, such as amethionine residue, at the N-terminus of the rPA amino-acid sequence.For example, the polypeptide may be SEQ ID NO: 6, or a fragment thereofcomprising the N-terminal methionine residue of SEQ ID NO: 6.

Also envisaged by the present invention is a kit, which may comprise oneor more of a polynucleotide, an expression vector, a host cell, and apolypeptide of the present invention.

Also provided by the present invention are antigenic compositions, suchas vaccine compositions, comprising a polypeptide according to thepresent invention.

The invention also provides methods of inducing an immune responseagainst infection by Bacillus anthracis comprising administering apolypeptide of the present invention or an antigenic composition of thepresent invention.

Also provided by the present invention is use of a polypeptide of thepresent invention for manufacture of a medicament for inducing an immuneresponse against infection by Bacillus anthracis.

In this regard, “inducing an immune response” may embrace protectingagainst infection by Bacillus anthracis. The protection conferred by themethod and/or use of the present invention may be 100%, or may be lessthan 100%. Preferably, “protecting against infection by Bacillusanthracis” provides protection against at least 50%, preferably at least70%, more preferably at least 80%, most preferably at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of Bacillus anthracisinfections. Preferably, “protecting against infection by Bacillusanthracis” provides a level of protection that is at least 50%,preferably at least 70%, more preferably at least 80%, most preferablyat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% effectiveagainst a Bacillus anthracis infection.

Furthermore, the term “protecting against infection” may embracepreventing infection and treating infection. In this regard, the term“preventing” includes reducing the severity/intensity of, or initiationof, a Bacillus anthracis infection. The term “treating” includespost-infection therapy and amelioration of a Bacillus anthracisinfection.

The antigenic composition may be administered by conventional routes,e.g. intravenous, subcutaneous, intraperitoneal, and mucosal routesusing methods well known in the art.

Typically, such antigenic compositions are prepared as injectables,either as liquid solutions or suspensions; solid forms suitable forsolution in, or suspension in, liquid prior to injection may also beprepared. The preparation may also be emulsified, or the peptideencapsulated in liposomes or microcapsules.

The active immunogenic ingredients are often mixed with excipients whichare pharmaceutically acceptable and compatible with the activeingredient. Suitable excipients are, for example, water, saline,dextrose, glycerol, ethanol, or the like and combinations thereof. Inaddition, if desired, the vaccine may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, pH buffering agents,and/or adjuvants which enhance the effectiveness of the vaccine.Examples of adjuvants which may be effective include but are not limitedto: aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine(thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637,referred to as nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphory loxy)-ethylamine (CGP 19835A, referred to as MTP-PE), andRIBI, which contains three components extracted from bacteria,monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton(MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion.

The active components may be formulated into the vaccine as neutral orsalt forms. Pharmaceutically acceptable salts include the acid additionsalts (formed with free amino groups of the peptide) and which areformed with inorganic acids such as, for example, hydrochloric orphosphoric acids, or with organic acids such as acetic, oxalic,tartaric, maleic, and the like. Salts formed with the free carboxylgroups may also be derived from inorganic bases such as, for example,sodium, potassium, ammonium, calcium, or ferric hydroxides, and suchorganic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol,histidine, procaine, and the like.

The antigenic compositions are conventionally administered parenterally,by injection, for example, either subcutaneously or intramuscularly.

The antigenic compositions are for administration in a manner compatiblewith the dosage formulation, and in such amount as will beprophylactically and/or therapeutically effective. The quantity to beadministered, which is generally in the range of 5 micrograms to 250micrograms of antigen per dose, preferably about 50-100 μg per dose,depends on the subject to be treated, capacity of the subject's immunesystem to synthesize antibodies, and the degree of protection desired.Precise amounts of the active ingredient that is required to beadministered may depend on the judgment of the practitioner and may beparticular to each subject.

The antigenic composition may be given in a single dose schedule, oroptionally in a multiple dose schedule. A multiple dose schedule is onein which a primary course of vaccination may be with 1-6 separate doses,followed by other doses given at subsequent time intervals required tomaintain and or reinforce the immune response, for example, at 1-4months for a second dose, and if needed, a subsequent dose(s) afterseveral months. The dosage regimen will also, at least in part, bedetermined by the need of the individual and be dependent upon thejudgment of the practitioner.

In addition, the antigenic composition containing the immunogenicantigen(s) may be administered in conjunction with otherimmunoregulatory agents, for example, immunoglobulins, as well asantibiotics.

Additional formulations which are suitable for other modes ofadministration include microcapsules, suppositories and, in some cases,oral formulations or formulations suitable for distribution as aerosols.For suppositories, traditional binders and carriers may include, forexample, polyalkylene glycols or triglycerides; such suppositories maybe formed from mixtures containing the active ingredient in the range of0.5% to 10%, preferably 1%-2%.

Oral formulations include such normally employed excipients as, forexample, pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharine, cellulose, magnesium carbonate, and thelike. These compositions take the form of solutions, suspensions,tablets, pills, capsules, sustained release formulations or powders andcontain 10%-95% of active ingredient, preferably 25%-70%.

In one embodiment the medicament may be administered intranasally(i.n.). An intranasal composition may be administered in droplet formhaving approximate diameters in the range of 100-5000 μm, which in termsof volume would have droplet sizes in the approximate range of 0.001-100μl.

Intranasal administration may be achieved by way of applying nasaldroplets or via a nasal spray. In the case of nasal droplets, thedroplets may typically have a diameter of approximately 1000-3000 μmand/or a volume of 1-25 μl, whereas in the case of a nasal spray, thedroplets may typically have a diameter of approximately 100-1000 μmand/or a volume of 0.001-1 μl.

It is possible that, following i.n. delivery of antibodies, theirpassage to the lungs may be facilitated by a reverse flow of mucosalsecretions.

In a different embodiment, the medicament may be delivered in an aerosolformulation. The aerosol formulation may take the form of a powder,suspension or solution.

The size of aerosol particles is one factor relevant to the deliverycapability of an aerosol. Thus, smaller particles may travel furtherdown the respiratory airway towards the alveoli than would largerparticles. In one embodiment, the aerosol particles have a diameterdistribution to facilitate delivery along the entire length of thebronchi, bronchioles, and alveoli. Alternatively, the particle sizedistribution may be selected to target a particular section of therespiratory airway, for example the alveoli.

The aerosol particles may be delivered by way of a nebulizer or nasalspray.

In the case of aerosol delivery of the medicament, the particles mayhave diameters in the approximate range of 0.1-50 μm, preferably 1-5 μm.

The aerosol formulation of the medicament of the present invention mayoptionally contain a propellant and/or surfactant.

By controlling the size of the droplets which are to be administered toa patient to within the defined range of the present invention, it ispossible to avoid/minimise inadvertent antigen delivery to the alveoliand thus avoid alveoli-associated pathological problems such asinflammation and fibrotic scarring of the lungs.

Intranasal vaccination engages both T and B cell mediated effectormechanisms in nasal and bronchus associated mucosal tissues, whichdiffer from other mucosae-associated lymphoid tissues.

Intranasal delivery of antigens allows targeting of the antigens tosubmucosal B cells of the respiratory system. These B cells are themajor local IgA-producing cells in mammals and intranasal deliveryfacilitates a rapid increase in IgA production by these cells againstthe anthrax antigens.

In one embodiment administration of the medicament comprising an anthraxantigen stimulates IgA antibody production, and the IgA antibody bindsto the anthrax antigen. In another embodiment, a mucosal and/or Th2immune response is stimulated.

In one embodiment, the vaccine composition comprises rPA proteinadsorbed to adjuvant. In one embodiment, the vaccine is delivered byintramuscular injection.

The invention also provides a vector having the sequence SEQ ID NO:4.

The invention also provides a vector as deposited under ECACC No:04061401.

The invention also provides a vector comprising a polynucleotideaccording to the present invention, as deposited under ECACC NO:04052501.

Referring to the Figures of the present application:—

FIG. 1 depicts HPA clone pMTL1015-cpg-rPA-synt—i.e. the modified rPAnucleic acid sequence fused to the cpg₂ leader in pMTL1015 (Tc^(R)).Manipulations required to generate this plasmid are as follows: (1)sub-clone synthetic ‘mature’ rPA sequence from sequence verified PCRproduct TOPO vector (i.e. without any leader) into HPA pET22bcpg vector;(2) sub-clone cpg-SynPA from pET22bcpgSynPA into pMTL1015.

FIG. 2 demonstrates SDS-PAGE of pMTL1015 clones expressing rPA after 16hr Shake-flask culture in phytone peptone-based Terrific Broth. The keyis as follows:—

1. Blank.

2. rPA Standard (DEV030IP; 100 μg/mL)

3. pMTL1015 vector only

4. pMTL1015-ompA-PA-wt

5. pMTL1015-cpg-PA-wt

6. pMTL1015-pelB-PA-wt

7. pMTL1015-ompA-PA-synt

8. pMTL1015-cpg-PA-synt

9. pMTL1015-pelB-PA-synt

10. Molecular weight markers

FIG. 3 shows a Western blot of pMTL1015 clones expressing rPA after 16hrs Shake-flask culture in phytone peptone-based Terrific Broth. The keyis as described above for FIG. 2.

FIG. 4 shows time course analysis by (A) SDS-PAGE and (B) Western Blotof samples from Shake-flask culture of E. coli (pMTL1015-cpg-PA-synt) inphytone peptone-based Terrific Broth. The negative control was E. coliRV308 (pMTL1015).

FIG. 5 shows growth curves of E. coli RV308 pMTL1015-ompA-PA-synt inphytone peptone-based Terrific Broth, Shake-flask culture, in baffledflasks (A and B) or non-baffled flasks (C).

FIG. 6 shows growth curves of E. coli RV308 pMTL1015-ompA-PA-wt (A andB) and pMTL1015-ompA-PA-synt [C and D) in Hy-soy based semi-definedmedium, Shake-flask culture.

FIG. 7 shows growth curves of E. coli RV308 pMTL1015-pelB-PA-wt (A andB) and pMTL1015-pelB-PA-synt [C and D) in Hy-soy based semi-definedmedium, Shake-flask culture.

FIG. 8 shows the growth curve of E. coli RV308 pMTL1015-cpg-PA-wt (A andB) and pMTL1015-cpg-PA-synt [C and D) in Hy-soy based semi-definedmedium, Shake-flask culture.

FIG. 9 shows SDS-PAGE (12.5% PHAST-GEL) of pMTL1015 clones expressingrPA in Shake-flask culture using Hy-soy based semi-defined medium. Thekey is as follows:—

1: Molecular Weight Markers

2: RV308 pMTL1015 ompA-PA-synt

3: RV308 pMTL1015 ompA-PA-wt

4: RV308 pMTL1015 pelB-PA-synt

5: RV308 pMTL1015 pelB-PA-wt

6: RV308 pMTL1015 cpg-PA-synt

7: RV308 pMTL1015 cpg-PA-wt

8: Reference DEV0303IP (100 μg/mL)

FIG. 10 shows growth of E. coli RV308 pMTL1015-cpg-PA-synt in productionmedium (Phytone peptone 12 g/L, Bacto yeast extract 60 g/L, glucose 25g/L, magnesium sulphate heptahydrate 2 g/L, K₂HPO₄ 12.54 g/L, KH₂PO₄2.31 g/L and tetracycline 1.5 mg/L, pH 7.0-7.2), with varying levels ofYeast Extract, as follows:—

A 1× yeast extract

B 1.5× yeast extract

C 2× yeast extract

D 2.5× yeast extract

FIG. 11 shows growth curves (A) and rPA production curves (B) for E.coli RV308 pMTL1015-cpg-PA-synt in production medium.

FIG. 12 is a flow chart showing the downstream processing steps forisolation of rPA.

FIG. 13 (A-D) shows the sequence alignment between SEQ ID NO:8—“DVC.Synthetic.rP” (i.e. the wild-type PA gene sequence, SEQ ID NO: 2,plus a 5′ codon encoding a methionine residue), and SEQ ID NO:7—“AP.PA.wt.Sequenc” (i.e. the modified rPA gene sequence of the presentinvention, SEQ ID NO: 1, plus a 5′ codon encoding a methionine residue).The sequence identity is 71.2%. (The consensus sequence is disclosed asSEQ ID NO: 106.)

The invention is now described by reference to the following Examples.

EXAMPLE 1 rPA Expression Systems

pMTL expression vector constructs were generated, directing expressionof either the wild-type PA gene sequence or the modified rPA genesequence, fused to either the Erwinia carotovora pelB or the pseudomonadcarboxypeptidase G₂ (cpg₂) leader sequences. The latter sequence isdescribed in European Patent 0 121 352, and has been shown to beefficiently processed in E. coli, directing soluble protein into theperiplasmic space. These rPA expression constructs were compared withDynport Vaccine Company (DVC)'s pET26b and Invitrogen pTrk vector-basedconstructs for evaluation.

Generation of novel rPA pMTL-based expression clones was as follows:

-   1. PCR amplification of the two PA-encoding nucleotide sequences    (wild type and modified) fused to both the pelB, ompA and cpg₂    leader sequences. This generated six rPA sequence options.-   2. Primary clones were constructed in a PCR product cloning vector    system (e.g. Invitrogen TA Cloning).-   3. Primary clones were authenticated by DNA sequence analysis of the    composite PA/leader sequences.-   4. The six rPA sequences were sub-cloned into pMTL1015 expression    vector and the recombinant plasmids were authenticated.-   5. Plasmid DNA derived from the four authenticated clones was used    to transform a protease-deficient expression strain, E. coli RV308    (ATCC 31608).

Strategy for Comparison of rPA Expression Systems

The six clones based on the pMTL expression plasmid were evaluated inshake-flask culture in the current production medium. The existing DVCproduction organism was used as a control. Growth conditions andinduction initiation/duration (where applicable) were standardised asmuch as possible to allow a true comparison of expression levels to bemade under the given experimental conditions. For example, a standardcell density was used for inoculation. Production levels were comparedby sampling cultures throughout growth and following chemical cell lysisusing BugBuster™ (Novagen) of harvested cells, by densitometric analysisof SDS-PAGE. Western blotting was used to confirm the identity of therPA protein band.

Media Selection Strategy

In addition to increasing the expression level of rPA by geneticmanipulation, the final yield of product was raised by growing culturesto a higher cell density using a medium containing higher nutrientlevels.

The strains showing the most promise in terms of rPA production levelsin the current production medium were examined further, initially inshake-flask culture, using a range of potential production media. Theanalytical techniques used by DVC (SDS-PAGE, RP-HPLC) were used toestimate product levels throughout growth and at harvest.

EXAMPLE 2 Shake-Flask Comparisons of rPA Expression EXAMPLE 2.1 PhytonePeptone-Based Terrific Broth

An experiment was performed to compare the expression of rPA by sixpMTL1015 clones in phytone peptone-based Terrific Broth usingshake-flask culture. Since previous work using the pMTL1015 expressionsystem at CAMR had shown that low oxygenation rates may favour productexpression, cultures were set up in both baffled flasks (highoxygenation) and non-baffled flasks (low oxygenation).

The 10 mL cultures prepared as primary seed cultures for the cellbanking were used to prepare seed cultures for this study. A 50 μLaliquot of the 10 mL culture was used to inoculate 50 mL of phytonepeptone-based Terrific Broth in 250 mL baffled flasks.

These seed cultures were incubated at 30° C. at 150 rpm for 17 h andthen used to inoculate duplicate 200 mL cultures of the same medium in1000 mL baffled flasks and single cultures of 250 mL in 500 mLnon-baffled flasks. The inoculum for each culture was calculated to givea starting OD₆₀₀ of 0.1-0.2. The cultures were incubated at 30° C. and150 rpm for 24 h. Samples (2.5 mL) were removed at 2 hourly intervalsfor rPA assay when the OD₆₀₀ reached 5-7. Samples were centrifuged at4,000 rpm in a Clandon T-52 bench top centrifuge for 15 min, thesupernatant decanted and the pellets stored frozen at −20° C. After 24 hgrowth, the duplicate baffled flask cultures were bulked and the cellmass harvested by centrifugation (Sorvall RC-3, 5000 rpm for 15 min) andthe cell paste stored frozen at −20° C.

FIGS. 2 and 3 show SDS-PAGE and Western blot analysis of the 16 hsamples from each of the pMTL1015 clones grown in phytone peptone-basedTerrific Broth under conditions of high oxygenation (baffled flasks)following treatment with BugBuster™. It can be seen that strong proteinbands are present at the expected rPA molecular mass following SDS-PAGEfor the three clones expressing the synthetic gene product, with weakerbands for those clones expressing the wild type gene (FIG. 2). Theamount of rPA present was estimated by comparison with the intensity ofthe rPA standard and confirmed by ELISA (Table 1). The Western blotanalysis demonstrated the presence of some immuno-reactive material atlower molecular weights than the rPA (FIG. 3). The amount of thismaterial relative to intact rPA was similar for all clones. It is notknown at present whether this represents proteolytic degradationproducts or truncated expression.

TABLE 1 Comparison of rPA expression of all 12 E. coli clones followinggrowth in phytone peptone-based Terrific Broth. rPA (μg/mL culture)OD₆₀₀ *Gel Clone Name E. coli host culture estimate ELISA 1 pET26b-PABL21 (DE3) 5.5 <100 24 2 pET26b-PA-synt BL21 (DE3) 3.6 <100 11 3pTrck-pelB-PA DH5a 9.7 <100 57 4 pTrck-ompA-PA DH5α 9.7 <100 74 5pMTL1015-pelB-PA-wt RV308 17.3 <100 58 6 pMTL1015-ompA-PA-wt RV308 19.7<100 135 7 pMTL1015-cpg-PA-wt RV308 20.4 <100 67 8 pMTL1015-pelB-PA-syntRV308 21.1 >>100  394 9 pMTL1015-cpg-PA-synt RV308 23.3 >>100  496 10pMTL1015-ompA-PA-synt RV308 25.3 >>100  476 11 pTrck-pelB-PA-synt DH5α8.2 >100 304 12 pTrck-ompA-PA-synt DH5α 11 >100 252 *estimate of rPAconcentration from SDS-PAGE by comparison with 100 μg/mL rPA standard.

Time course samples from the pMTL1015-cpg-PA-synt clone were analysed bySDS-PAGE (Phast-gel), Western Blot (FIG. 4) and ELISA (Table 2) todetermine; (a) the point at which rPA expression was maximal, and (b)whether prolonged incubation resulted in loss of product due toproteolytic activity. It can be seen that rPA expression by ELISA wasoptimal after 14-16 h incubation and did not change appreciably withfurther incubation up to 24 h (Table 2). Western blot analysis (FIG. 4)showed that the level of lower molecular weight immuno-reactive materialrelative to intact rPA did not change significantly with extendedincubation time.

TABLE 2 Time course analysis by ELISA of samples from shake flaskculture of E. coli (pMTL1015-cpg-PA-synt) in phytone peptone basedTerrific Broth. Time (h) rPA (μg/mL culture) 10 107 12 313 14 507 16 53218 488 20 491 22 487 24 525

FIG. 5 shows the growth curves obtained for E. coli RV308(pMTL1015-ompA-PA-synt) when grown in phytone peptone-based TerrificBroth using baffled (high oxygenation) and non-baffled (low oxygenation)flasks. It can be seen that growth was substantially better in thebaffled flasks. Cultures grown in non-baffled flasks reached a muchlower final cell density compared with the baffled flask cultures. rPAexpression was considerably lower in the cultures grown in non-baffledflasks (data not shown).

EXAMPLE 2.2 Hy-Soy-Based Semi-Defined Medium

The experiment described above (Example 2.1) was repeated using Hy-Soybased semi-defined medium in baffled flasks only. The growth curves(FIGS. 6, 7 & 8) show that lower growth rates and final cell densitieswere obtained in this medium compared to Terrific Broth and a lag phaseof up to 8 h was obtained for the clones expressing the synthetic gene.rPA expression levels were generally lower than observed in phytonepeptone-based Terrific Broth; however, a similar pattern of superiorexpression levels with clones expressing the synthetic gene comparedwith the wild-type gene was observed by SDS-PAGE (FIG. 9) and ELISA(Table 3).

TABLE 3 Comparison of rPA expression of E. coli pMTL1015 clonesfollowing growth in Hy-soy-based semi-defined medium. rPA (μg/mLculture) E. coli Sample *Gel Clone Name host time (h) estimate ELISA 5pMTL1015-pelB-PA-wt RV308 16 <100 50 6 pMTL1015-ompA-PA-wt RV308 16 <10064 7 pMTL1015-cpg-PA-wt RV308 16 <100 56 8 pMTL1015-pelB-PA-synt RV30820 >100 224 9 pMTL1015-cpg-PA-synt RV308 20 >100 170 10pMTL1015-ompA-PA-synt RV308 20 >100 189 *estimate of rPA concentrationfrom SDS-PAGE by comparison with 100 μg/mL rPA standard

EXAMPLE 3 Fermenter Level Comparisons of rPA Expression

Evaluation of the following four down-selected clones in fermenterculture was continued:

E. coli RV308 pMTL1015-cpg-PA-synt

E. coli RV308 pMTL1015-ompA-PA-synt

E. coli RV308 pMTL1015-ompA-PA-wt

E. coli DH5 pTrcK-pelB-PA-synt

Medium Selection

8 L fermentations were performed in each medium under conditions aspreviously described with DOT and pH control.

The growth curves obtained were similar to those seen previously withthe same media (see FIG. 10), but the rPA yield from production medium(Run No: PRECRV0034: Table 4) was 2500 μg/mL by ELISA. This culture wasfed with 80 mL of 50% glucose solution prior to glucose depletion in theculture. A growth curve for PRECRV0034 showing rPA production can beseen in FIG. 11, but data does not indicate whether the yield hasreached a maximum when the culture was harvested.

In order to determine whether the improved rPA yields obtained for E.coli RV308 pMTL1015-cpg-PA-synt when cultured in production alsooccurred in the other two down-selected pMTL1015 clones, parallelfermentations were set up for all three strains under these conditions.However, the 80 mL of 50% glucose fed to the previous cultures wasincluded from the start, raising the initial glucose concentration to 25g/L.

E. coli RV308 pMTL1015-cpg-PA-synt again gave a yield of 2500 μg/mL andE. coli RV308 pMTL1015-ompA-PA-synt yielded 2000 μg/mL (see PRECRV0038and 0037, Table 4).

E. coli DH5α pTrcK-pelB-PA-synt was grown in PPTBgIy at 8 L scale (seeTable 4, PRECDH0013) with the exception that the OD₆₀₀ at induction wasraised to 15. The rPA yield was not improved significantly over previousresults although more biomass was produced with a higher final OD₆₀₀ of26 at four hours post induction with IPTG.

Effect of Growth Temperature

Previous development programs incorporating the E. coli RV308pMTL1015-cpg-host/vector system have indicated that expression ofproduct is most efficient at temperatures between 25 and 30° C.

Assessment of the effect on yield and product stability of growth atlower temperatures of E. coli RV308 pMTL1015-cpg-PA-synt was made byculturing the strain in production medium, under conditions describedabove, at 30, 28 and 25° C. (see Table 4, PRECRV0039, 0040 and 0041respectively).

The yields from production at the lower temperatures were lower thanwhen grown at 30° C. The quality of the material produced did notimprove with the reduction in temperature, with little or no reductionin minor impurity bands on SDS-PAGE/Western Blot.

Effect of Antibiotic Concentration

As a confirmation of the stability of the plasmid under reducedantibiotic selective pressure, E. coli RV308 pMTL1015-cpg-PA-synt wascultured in production medium, under conditions described above, withvarying tetracycline concentration levels in the medium (see Table 4,PRECRV0042-0044). The tetracycline concentrations were 15 μg/mL (100%),1.5 μg/mL (10%) and 0. The 2° seed cultures contained 15, 1.5 and 15μg/mL respectively. Thus the fermenter with no added antibiotic reliedon carryover from the secondary seed to supply any selective pressure,assuming no degradation of the tetracycline during the seed growth. Thevolume of seed transferred to the fermenter was 124 mL giving a nominal0.23 μg/mL tetracycline in the fermentation medium at inoculation.

The yields in terms of final OD₆₀₀ and biomass were within the expectedrange, but the yield of rPA was slightly lower than expected for the 15μg/mL control. The levels for the reduced antibiotic cultures wereslightly higher. The stability of the pMTL1015-cpg-PA-synt plasmid wasconfirmed by tooth-picking final fermentation sample colony isolatesonto selective (L-agar with 15 μg/mL tetracycline) and non-selectivemedia. The results of 100, 98 and 96% growth on selective medium for 15,1.5 and 0 μg/mL tetracycline fermentations respectively, indicate goodstability under the conditions used. The viable count results forPPTBgIuc2.5xYE fermentations are in the 2×10¹⁰-5×10¹⁰ cfu/mL range.

TABLE 4 Summary Table of fermentations. SDS- Seed Fermenter PAGE ELISARun no. Clone Medium (No.) OD₆₀₀ OD₆₀₀ (mg/L) (mg/L) PRECRV pMTL1015-PPTBgluc 12.6 34.8 100+   320 0031 cpg-PA-synt PRECRV pMTL1015- PPTBgluc13.0 44.2* 100*    84* 0032 cpg-PA-synt YEx1.5 PRECRV pMTL1015- PPTBgluc14.7 59.8 500++ 1630 0033 cpg-PA-synt YEx2 PRECRV pMTL1015- PPTBgluc14.2 71.6 500++ 2500 0034 cpg-PA-synt YEx2.5 PRECDH pTrcK-pelB- PPTBgly2.67 26.2 100++ 360-465 0013 PA-synt PRECRV pMTL1015- PPTBgluc 14.843.9* <100*    90* 0036 ompA-PA-wt YEx2.5 PRECRV pMTL1015- PPTBgluc 8.756.6 500++ 2000 0037 ompA-PA- YEx2.5 synt PRECRV pMTL1015- PPTBgluc 14.762.6 500++ 2500 0038 cpg-PA-synt YEx2.5 PRECRV pMTL1015- PPTBgluc 15.662.4 500++ 2300 0039 cpg-PA-synt YEx2.5 PRECRV pMTL1015- PPTBgluc 15.662.2 500++ 1600 0040 cpg-PA-synt YEx2.5 PRECRV pMTL1015- PPTBgluc 15.663.6 500++ 1500 0041 cpg-PA-synt YEx2.5 PRECRV pMTL1015- PPTBgluc 12.962.4 500++ 1720 0042 cpg-PA-synt YEx2.5 PRECRV pMTL1015- PPTBgluc 13.266.4 500++ 1800 0043 cpg-PA-synt YEx2.5 PRECRV pMTL1015- PPTBgluc 12.963.8 500++ 2120 0044 cpg-PA-synt YEx2.5 PPTB—Phytone Peptone-basedTerrific Broth; gluc—glucose; YE—Yeast Extract

Selection of Production Strain

The results obtained to date for the clones investigated after theinitial down-selection have shown that of the four, E. coli RV308pMTL1015-cpg-PA-synt has, in most cases, shown the highest yield whencompared with the other pMTL1015 clones under equivalent conditions.

The largest proportion of information generated has been from the E.coli RV308 pMTL1015-cpg-PA-synt clone, for both fermentation and DSPdevelopment and with yields in the 1.5-2.5 mg/mL range when productionmedium has been used. This has allowed the present applicant to selectthis clone as their preferred production organism for all future work.

Table 5 shows a summary of all cultures grown to date in productionmedium. The figures indicate that although 2500 μg/mL is achievable, amore realistic value for the yield is 2000 μg/mL.

TABLE 5 Comparison of rPA levels relative to cell wet weight and opticaldensity at harvest for cultures of E. coli RV308 pMTL1015-cpg-PA-syntcontaining 2.5x yeast extract. Cell rPA mg rPA/g Culture Weight Yield mgrPA/ wet Run No. OD₆₀₀ (g/L) (mg/L) OD unit weight PRECRV0030 69.8 85.81900 27.2 22.1 PRECRV0034 71.6 102 2500 34.9 24.5 PRECRV0038 62.6 86.52500 39.9 28.9 PRECRV0039 62.4 77.5 2300 36.9 29.7 PRECRV0040 62.2 80.41600 25.7 19.9 PRECRV0041 63.6 98.5 1500 23.5 15.2 PRECRV0042 62.4 89.11720 27.6 19.3 PRECRV0043 66.4 88.8 1800 27.1 20.3 PRECRV0044 63.8 83.92120 33.2 25.3 MEAN 65.0 88.1 1993 30.7 22.8

EXAMPLE 4 Upstream Process for rPA Production

Seed Banks for Clone of Interest—Clone pMTL1015-cpg-PA-synt Transformedinto E. coli RV308 (ATCC 31608).

After sequence confirmation, a research seed bank was prepared by growthunder selective pressure of tetracycline (15 mg/L) in soy peptone basedL-broth (Phytone peptone 15 g/L, Bacto yeast extract 5 g/L, NaCl 5 g/L,pH 6.8-7.0). A single colony from a nutrient agar plate withtetracycline was inoculated into 100 mL medium in 500 mL baffled shakeflasks and incubated at 30° C. and 150 rpm in a shaking incubator untilOD₆₀₀ reached 1.5. The culture was then mixed with sterile 50% glycerolin growth medium (see above) to give a final glycerol concentration of10%, and stored frozen at −80° C. as 1 mL aliquots in 1.8 mL cryovials.

A working research cell bank (WRCB) of 250 vials was prepared from aboveseed bank using the same conditions and medium for growth, however 250μL of thawed vial contents were inoculated into 200 mL medium in 1000 mLbaffled shake flasks.

Primary Seed Culture.

1 vial of WRCB was thawed and 100 μL inoculated into 10 mL soy peptonebased L-Broth (see above) containing tetracycline at 15 mg/L in a 25 mLuniversal bottle, incubated at 30° C. with shaking at 150 rpm for 7-9hours. This was a recovery step to ensure that the organism is viableand to give a more consistent seed production process. The final OD₆₀₀of this step was 0.7-1.0.

Secondary Seed Culture.

This step produces the inoculum for the fermentation, and with areasonably sized shaking incubator is capable of producing inoculasufficient for 5-250 L cultures in shake flasks. At 50 L scale, 200 μLof primary seed was inoculated into 200 mL of production medium in eachof 5×1000 mL baffled shake flasks. The cultures were incubated withshaking at 150 rpm and 30° C. for 11-12 hours giving a final OD₆₀₀ of13-16.

To prevent precipitation and caramelisation of some components duringsterilisation by autoclaving, production medium is prepared bysterilising the complex component as a bulk and then adding the glucose,phosphate, magnesium and tetracycline aseptically as sterile solutionswhen the temperature of the components has fallen to lower than 25° C.

Production Fermentation

The seed cultures were then bulked and a volume sufficient to give astarting OD₆₀₀ of 0.2 in the fermenter was inoculated into 50 Lproduction medium (see above) in a 72 L Applikon stirred tank fermenter.The complex medium components were sterilised, as a 40 L bulk, in situat 121-123° C. for 30 minutes, cooled to below 25° C. and thensupplemented with the remaining components to bring the total volume to50 L.

The culture was then grown as a batch at a temperature of 30(±0.5)° C.,pH 7.0 controlled by addition of sodium hydroxide and phosphoric acid.Dissolved oxygen tension was maintained at >40% by cascade step controlof the following parameters: agitation (200-800 rpm), backpressure (3-7psi), sparged airflow (25-50 Lpm) and oxygen supplementation (0-20 Lpm),in the order described.

When growth had ceased (12-14 hours), as measured by OD (OD₆₀₀ 60-65),the culture was chilled to below 15° C. and harvested by batchcentrifugation (Sorvall RC-3B, H6000A rotor, 5000 rpm for 15 minutes).The harvested cell paste was stored at −20° C. until required fordownstream processing. Product expression was assessed by ELISA assayfrom samples removed hourly from the culture.

EXAMPLE 5 Downstream Processing Steps

Cell Breakage

Approximately 4.5 kg of frozen cell paste harvest were suspended into asmooth paste with, initially, a minimum volume of 20 mM tris/1 mM EDTApH 8.5. Further buffer was added to give an overall suspended volume of16 L.

The suspended cells were broken by passing twice through an ‘APV Gaulin’high-pressure homogeniser at a pressure of 7000 psi. The homogenate wasthen centrifuged for 1 hour at 5000 rpm in a ‘Sorval’ RC3 centrifuge.The pellet was discarded, and the supernatant (16 L approx) wasretained.

Diafiltration

The centrifuged homogenate was diafiltered with 3 times its volume ofpurified water using a ‘Millipore Pellicon’ concentrator fitted with two‘Pall’ OS030F07 0.5 m² ‘Centrasette 2 Omega’ suspended screen channel 30kDa membranes. The concentrator was operated at a flow-rate of 17 L/minwith a trans-membrane pressure of 1.6 Bar. The pH was adjusted to 8.0and the conductivity to 2 mS/cm.

Anion Exchange and Chromatography

A 25 cm diameter chromatography column was packed with 5 L of ‘Amersham’‘Q-Sepharose Fast Flow’ anion exchanger to give a bed height of 10 cm.An industrial UV monitor was then connected to the effluent line. Thecolumn was operated at a flow-rate of 330 mL/min throughout. The packedcolumn was washed with 10 L of water, then 5 L of 0.5 M sodiumhydroxide, followed by purified water. 10 L of 0.5 M tris, pH 8.0, waspumped, and the column was then equilibrated with start buffer (20 mMtris, pH 8.0).

The diafiltrate was loaded, and then the loaded column was washed tobaseline resolution with start buffer. The bound rPA was eluted withincreasing salt steps of 10, 20, and 65 mM sodium chloride in startbuffer, and the eluted peaks were collected in separate appropriatelysized vessels. The eluates were assayed by SDS-PAGE and SEC-HPLC, andthe fractions containing rPA at a purity of >40% were retained.

The column was regenerated by passing sequentially 10 L of 2 M sodiumchloride, followed by 10 L of 1 M sodium acetate, 10 L of 0.5 M sodiumhydroxide, then 10 L of 50 mM sodium hydroxide for storage.

Hydrophobic Charge Induction Chromatography

A 30 cm diameter column connected to UV monitor was packed with 20 L of‘Ciphergen’ ‘MEP HyperCel’ at a flow rate of 7 L /min. Once packed, allfurther steps were performed at 800 mL/min. The column was washed with 5L of 1 M sodium hydroxide with a contact time of no more than 40 min.The column was then washed with water, and then equilibrated with 20 Lloading buffer (50 mM tris, pH 8.0). The Q pool (i.e. the pool from theprevious Q chromatography step) was loaded, the column was washed withloading buffer to baseline, then the bound rPA was eluted with purifiedwater. The collected product was assayed by SDS-PAGE and SEC-HPLC. TheMEP pool (i.e. the pool from the MEP Hypercel column) was filteredthrough a 0.22 μm, 2000 cm² Pall ‘Posidyne’ filter. The column wasregenerated with 10 L of 1 M sodium hydroxide, washed with purifiedwater, and then stored in 0.2 μm filtered 50 mM sodium hydroxide.

Diafiltration and Formulation

The purified rPA was diafiltered using a ‘Pall Centramate’ medium screen‘Omega’ 50 kDa cartridge (part No. OS0350C12, 0.093 m²). A flow-rate of800 mL/min, and a trans-membrane pressure of 1.6 Bar were used. Thediafiltration was performed versus 5 L of formulation buffer; 25 mMsodium phosphate, 150 mM sodium chloride, pH 8.0. A further filtrationwas performed using a 0.22 μm Pall ‘Posidyne’ filter of 5000 cm² area,and then the final product was dispensed into appropriate vials.

EXAMPLE 6 Construction of Variant Synthetic rPA Gene Constructs

The variant sequences set out in SEQ ID NOS: 9-105 are synthesized usingsolid phase chemical synthesis using nucleoside phosphoramidites. Thisis a well-established method in the field (see Brown T, Brown D J S.1991. in Oligonucleotides and Analogues. A Practical Approach, ed. FEckstein, pp. 1-24. Oxford: IRL). Typically, the synthetic genesequences are constructed from a number of oligonucletide sequences(40-80 bp in length) that have been generated using this chemicalsynthesis methodology. These oligonucleotide sequences represent bothstrands of the gene sequence and have their termini designed such that,post hybridization of complementary oligonucleotide pairs, the doublestranded elements are bound by unique complementary overhangingsequences that enable their correct ordered assembly to generate the rPAgene “sub-fragment” sequences, typically ˜500 base pairs in length. Thisis performed by mixing the hybridized oligonucleotide pairs with anappropriately cleaved plasmid vector (for example, a PUC or InvitrogenTOPO vector) with the addition of bacteriophage T4 ligase; both plasmidand rPA sub-fragments having compatible restriction site termini. Theseligation products are used to transform competent E.coli host cells,generating E.coli clones for screening. Screening of clones is carriedout using restriction enzyme analysis of isolated plasmid DNA, andselected clones authenticated by DNA sequence analysis of the clonedinsert of the plasmid.

The final rPA-encoding gene product is then assembled via the isolationof the plasmid-borne “sub-fragment” DNA segments as specifically boundrestriction fragments, which are then ligated together with anappropriately cleaved plasmid vector (for example, a PUC or InvitrogenTOPO vector) and the ligation mixture used to transform competent E.colicells. The E.coli clones generated are then screened by restrictionanalysis and the entire cloned rPA-encoding gene sequence verified byDNA sequence analysis.

The synthetic rPA-encoding gene sequence may be generated with orwithout a 5′-leader sequence DNA moiety. If generated without a leadersequence, a unique MscI restriction site is engineered at the 5′-end ofthe coding sequence as this will enable “in-frame” cloning to someleader sequences (e.g., ompA, pelB) which already reside in somecommercially available E.coli expression plasmids. More typically, thechoice of a larger range of leader sequences (e.gs, cpg2, ompA, pelB,phoA, ompT, lamb, omp F, beta lactamase, Staphylococcus aureus ProteinA, Bacillus subtilus endoglucanase, murine RNAase, human growth hormone,enterotoxins ST-II, LT-A and LT-B) is incorporated in the originaldesign and DNA synthesis strategy. This way, the leader sequence-rPA“cassette” is generated as a single genetic element that can then besub-cloned into a variety of E.coli expression vectors for production ofthe rPA polypeptide product. By virtue of engineering a unique MscIrestriction site at the leader sequence-rPA-encoding gene sequence,different leader sequences may be substituted via simple sub-cloningprocedures (Sambrook, J., Fritsch, E. F., Maniatis, T. MolecularCloning: a Laboratory Manual, 2^(nd) Ed. 1989. Cold Spring HarbourLaboratory Press).

EXAMPLE 7 Expression of Variant Synthetic rPA Gene Constructs

The rPA gene encoding sequences depicted in SEQ ID NOs: 9 to 105 arecloned as leader sequence-rPA sequence “cassettes” into E.coliexpression plasmids.

Cloning of these leader sequence-rPA sequence “cassettes” is facilitatedby virtue of unique restriction sites engineered at the 5′- and 3′-endsfor compatibility with the E.coli expression vector of choice.Typically, this would be either a NdeI or NcoI restriction site at the5-end as this facilitates optimal positioning of the ATG initiation(metheionine) codon to the vector borne ribosome binding site (RBS) toenable efficient translation initiation of the RNA message originatingfrom the expression vector moiety of the constructs. Restriction enzymesites at the 3′-ends of the leader sequence-rPA sequence “cassettes” arevariable and engineered to be compatible with choice of E.coliexpression vector. The choice of available E.coli expression vectors isvaried and examples include: pMTL series of vectors (proprietary toHPA), pET series (Novagen), pRSET, pET and pBAD series (Invitrogen), pQEseries (Qiagen), pMaI series (New England Biolabs), pPROTet series(Clontech), pGEX series (GE Healthcare), pEK/LIC series (EMDBiosciences), pIVEX series for cell-free expression (Roche) andbacteriophage Lambda gt11 and Lambda ZAP expression vectors(Stratagene).

Expression of the rPA polypeptides elaborated from rPA-coding sequencesSEQ ID Nos 9 to 105 is conducted in shake flasks or in fermenterbioreactors as described in Examples 2 and 3. The latter of these (i.e.the fermenter) attains levels of periplasmically located rPA polypeptideproduct at levels typically exceeding 2 grams per litre. Particularlygood levels of rPA polypeptide (in the range 2.1-2.4 grams per litre)are achieved using SEQ ID NOs: 36, 46, 56, 66, 76, 86 and 96. SEQ ID NO:36, for example, achieves a typical yield of at least 2.2 grams perlitre.

Purification of the rPA polypeptide is achieved using the methodsdescribed in Example 5.

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1-27. (canceled)
 28. A method for producing rPA comprising expressing apolynucleotide having at least 90% sequence identity to SEQ ID NO: 36,wherein the polynucleotide is an rPA polynucleotide, with the provisothat the polynucleotide does not comprise the nucleic acid sequence ofSEQ ID NO:
 1. 29. A method according to claim 28, wherein saidpolynucleotide is expressed in a host cell.
 30. A method according toclaim 28, wherein said polynucleotide is DNA, and is optionally firsttranscribed into RNA in vitro and the RNA is then translated in a hostcell.
 31. A method according to claim 29, wherein the host cell is an E.coli host cell.
 32. A method according to claim 31, wherein the E. colihost cell is E. coli RV308 (ATCC No: 31608).
 33. A method according toclaim 28, comprising expressing rPA from an expression vector comprisingthe polynucleotide.
 34. A method according to claim 33, wherein theexpression vector comprises the cpg leader sequence encoded by SEQ IDNO:
 3. 35. A method according to claim 33, wherein the vector is plasmidpMTL1015, encoded by SEQ ID NO:
 4. 36. A method according to claim 33,wherein the vector is expressed in E. coli RV308 (ATCC No: 31608).
 37. Amethod according to claim 33, further comprising initial steps oftransforming the expression vector into a host cell, and culturing thetransformed host cell in a growth medium.
 38. A method according toclaim 37, wherein said growth medium is substantially free of animalproducts.
 39. A method according to claim 37, comprising culturing thetransformed host cells at a reduced temperature.
 40. A method accordingto claim 37, further comprising harvesting the host cell.
 41. A methodaccording to claim 40, further comprising extracting rPA from the hostcell.
 42. A method according to claim 41, further comprising aseparation step.
 43. A method according to claim 42, wherein theseparation step is selected from one or more chromatography steps, andone or more filtration steps.
 44. A method according to claim 43,wherein at least one of said filtration step(s) is a diafiltration step.45. A method according to claim 43, wherein at least one of saidchromatography step(s) is selected from an ion-exchange chromatographystep and a hydrophobic charge chromatography step.
 46. A methodaccording to claim 29, comprising: (a) obtaining host cells that expressthe polynucleotide or an expression vector comprising thepolynucleotide; (b) extracting the expressed rPA from the host cells;(c) subjecting the extracted rPA to a diafiltration step; (d) subjectingthe diafiltered rPA to at least one chromatography step selected fromanion exchange and hydrophobic charge chromatography; and (e) carryingout a further diafiltration step.
 47. (canceled)